gene expression profiling in breast carcinoma

GENE EXPRESSION PROFILING IN BREAST CARCINOMADr Partha Ghosh2nd year PGT, General SurgeryN.R.S Medical college

What is gene expression profiling?Cancer is a disease characterized by uncontrolled

cell growth and proliferation. For cancer to develop,  genes regulating cell growth and differentiation must be altered; these mutations are then maintained through subsequent cell divisions and are thus present in all cancerous cells. Gene expression profiling is a technique used in molecular biology to query the expression of thousands of genes simultaneously. In the context of cancer, gene expression profiling has been used to more accurately classify tumours. The information derived from gene expression profiling often has an impact on predicting the patient’s clinical outcome.

Tumor biology

IHC mRNA profiling Histology TNM Staging

Breast Cancer SubtypesBreast cancer is classified into clinical

subtypes based upon receptor expressionThese subtypes dictate possible therapeutic

options and vary in their prognosis◦ Luminal: derived from the luminal cells

TYPE A:ER+, PR+,HER-2- TYPE B:ER+,PR+,HER-2+ Can use hormonal therapy Less aggressive

◦ Basal: derived from myoepithelial cells ER-, PR-,HER-2-,ck 5/6+ or HER -1+ No specific target for therapies More aggressive

◦ HER2-enriched More aggressive

Luminal A

Luminal B

Claudin-Low

HER2-enriched

Basal

Luminal and Basal Characteristics

BasalLow ERLow HER2High CK5/6c-KIT higherHigh EGFRHigh p53

mutationHigh p53 proteinHigh cyclin EVery high

vimentin

LuminalHigh ERHigher HER2Low CK5/6Low c-KITLow EGFRLow p53

mutationLow p53 proteinLow cyclin ELow vimentin

Basement membraneMyoepithelial Cells BasalLuminal Cells Luminal

What is HER 2/neuOther names

◦Receptor tyrosine-protein kinase erbB-2

◦ CD340 ◦ proto-oncogene Neu◦ ERBB2 (human)Located in chromosome 17 (17q12)Rodent glioblastoma cell line a neural

tumour

Cont….Plasma membrane bound receptor

tyrosine kinasePositive in 15-30% cases of breast

carcinomaAssociated with increase disease

recurrence and poor prognosisTested by-

◦Immunohistochemistry ◦FISH◦Serum HER2 by ELISA- Trastuzumab

response

Cont…Drugs targeting HER2

Trastuzumab- p27 halts cell proliferatonPertuzumab- prevent dimerisation of HER2 and HER3NeuVax- Direct killer T cells to destroy HER2 +ve cancer cells

Tamoxifen resistant breast caThe expression of HER2 is

regulated by signaling through estrogen receptors.

Normally tamoxifen down regulate HER2

Ratio of coactivator AIB-3 exceed that of corepressor PAX2 HER2 expression in presence of tamoxifen

Algorithm for breast cancer subtypes

All cases

ER - PR -ER+ or PR +

HER2 - HER2 + HER2 +

HER2 -

EGFR - CK5/6 – EGFR + or CK5/6 +

Unclassified Basal Like Luminal B Luminal AHER2+/ER-

Luminal ALuminal B

HER2+Basal-like

Intrinsic Breast CancerSubtypes described by

Perou et al.

Express ↑ amountsOf luminal cyto-Keratins & geneticMarkers of luminalEpithelial cells ofNormal tissue

Express ↑ levels of EGFR, c-kit, & growth factors like hepatocyte growth factor and IGF

Mammaprint: Development of the 70-Gene Signature DNA microarray analysis of 78 breast primary tumors

(untreated) Pts were <55 years of age with T1-2/N0 disease Pts selected based on outcome: Distant metastases

within 5 years

Statistical analysis, “supervised classification,” identified 231 genes correlated with disease outcome Top 70 genes selected

Genes that regulate cell cycle, invasion, metastasis, & angiogenesis

Patients categorized as “good prognosis” or “poor prognosis.”

Found to be a better predictor of distant metastases within 5 years than all clinical variables in this study

Odds ratio (distant metastases): poor to good prognosis groups = 15

Does 70-gene Signature have Independent Prognostic Value?

Gene signature adds independent prognostic information to that provided by various risk classifications

The signature remained a statistically significant prognostic factor for time to distant metastases & OS even after adjustment for various risk classifications (HR 2.15 & 2.15, respectively)

The 21-gene Recurrence Score: Oncotype DX

Oncotype Dx: The 21-Gene Assay Designed to quantify the risk of distant recurrence in

patients with LN(-), ER(+) tumors receiving tamoxifen RT-PCR was used to quantify gene expression from

fixed, parafin-embedded tumor tissue 250 candidate genes selected based on published

literature, genomic databases, & experiments based on DNA arrays on fresh-frozen tissue

Analyzed data from 3 independent studies (447 patients) including tamoxifen-only arm of NSABP trial B-20 to test relation b/w 250 genes and recurrence of breast cancer

From these studies, 16 genes (+5 reference genes) were selected that correlated with proliferation and endocrine response

Levels of Gene Expression Determine Recurrence Score

21-gene assay = 16 outcome-related genes + 5 reference genes

Higher expression levels of“favorable” genes = ↓ RS

Higher expression levels of“unfavorable” genes = ↑ RS

A risk score is calculated from 0 -100Cutoff points chosen based onResults of NSABP trial B-20

Prospective Validation of Oncotype DX:TAILORX Trial

Low RS:HormonalTherapy

High RS:Chemo +HormonalTherapy

Hormonal Therapy Chemo + Hormonal

11,248 ER+/LN- patients

TN VS BASAL SubtypesTriple negative

◦ ER,PR,HER2 receptor negative

Basal Subtypes◦ ER,PR,HER2

receptor negative◦ Over expression

of breast basal cell epithelium gene

◦ EGFR+,CK5/6+

Basal like subtypes of breast cancerNo proven therapeutic target

ER,PR negative-cannot use tamoxifen or anti estrogen

HER2 negative-cannot use Herceptin

Targeted therapy ER + ER-

Hormonal Herceptin EGFR BRCA 1 C-kit

Therapy Geftinib, erlotinib, DNA damage Imatinib

lapatinib PARP inh

Luminal A

Luminal B HER 2 + Basal

Like

Potential treatment options in TNBC

Taxane and Anthracycline Based TherapyTypical regimens:

AC-T: doxorubicin plus cyclophosphamide every 2 weeks for four cycles followed by docetaxel every 2 weeks for 4 cycles

TAC: docetaxel, doxorubicin, and cyclophosphamide every 3 weeks for 6 cycles

Dense dosing is better

Platinum AgentsPlatinum agents can bind to DNA

and cause cross-linking to occur cell death

Cisplatin, carboplatin and oxaplatin are approved for some types of cancers and are being studied as treatments for TNBC

PARP InhibitorsPARP: poly ADP ribose

polymerase◦Involved in DNA repair by detecting

single-strand breaks◦Can be activated in cells with

damaged DNASeveral types of cancer are more

dependent on PARP, so it can be a good therapeutic target

PARP inhibitors prevent breaks from being repaired, causing cell death

Anti-EGFREGFR is overexpressed in 45-70% of TNBC Cetuximab is an anti-EGFR antibody used to

treat metastatic cancer◦Breast cancer patients with metastatic disease

respond twice as well when Cetuximab is addedOther treatments include tyrosine kinase

inhibitors (erlotinib, gefitinib)◦Gefitinib is the only one currently approved for

breast cancer, but the others are in clinical trialsInhibits an important signaling pathway and

provides a specific target!

Angiogenesis in CancerAngiogenesis: formation of new

blood vessels. ◦Tumors need blood vessels to grow and

spread.Angiogenesis inhibitors prevent the

formation of new blood vessels, thereby stopping or slowing the growth or spread of tumors.

Anti-AngiogenesisBevacizumab (Avastin)

◦ Monoclonal antibody to VEGF◦ Improves survival in breast cancer patients with

combined with Taxol◦ Approved for metastatic breast cancer but benefit

isn’t subtype specific – this has since been revoked because it slowed progression but didn’t extend length or quality of life and had many adverse effects

Metronomic chemotherapy: repeated, low, less than toxic doses can destroy endothelial cells and prevent angiogenesis, slowing tumor growth – works in clinical trials

Androgen ReceptorNuclear receptor activated by binding

testosterone or dihydrotestosterone◦ Closely related to PR

Expressed in 75% of breast cancer and 10-20% of TNBC◦ TNBC that express AR are molecularly similar to

prostate cancer and could potentially be treated similarly.

Bicalutamine: anti-androgen used to treat prostate cancer

17-DMAG: semi-synthetic antibiotic derivative, has shown promise in clinical trials

Enzalutamide: androgen agonist used to treat prostate cancer; is in Phase II for TNBC

RTK InhibitorsSuninitib (Sutent)

◦Multiple-target RTK inhibitor All PDGFRs and VEGFRs KIT (CD17) which drives the majority of all GI

stromal tumors & several others

Imatinib (Gleevec)◦Prevents phosphorolation of BCR-Abl,

inhibiting signaling pathways necessary for cancer cell growth BCR-Abl: Exists only in cancer cells! Worked in vitro; no effect on metastatic

breast cancer patients in Phase II

Src Tyrosine KinaseSrc is overexpressed in breast

cancerDasatinib: multiple tyrosine kinase

inhibitor approved for CML◦Possible efficacy in breast cancer -

small effect seen in Phase II◦In vitro: basal breast cancer cells were

more sensitive!Several others in trials also seem to

have promising preclinical activity

mTORCell cycle regulator and a downstream

effector in the PI3K/PTEN/AKT pathwayPTEN is often mutated in TNBC, leading

to increased AKT and mTOR activationEverolimus and temsirolimus block mTOR

function and inhibit proliferation◦Everolimus is approved for some types of

cancers - currently in clinical trials for TNBC in combination with chemotherapy

◦Temsirolimus is approved for renal cell carcinoma and completed a Phase II trial with promising results

ConclusionsGene signatures augment current

clinicopathological variables in assessing risk of recurrence

Gene expression profiles may be both prognostic and predictive for patients with early breast cancer

NCCN guidelines suggest that Oncotype DX is an option for risk evaluation in 0.6-1 cm tumors with unfavorable characteristics or in >1 cm LN-, ER+/HER2 negative tumors

NCCN guidelines include Oncotype DX® testing in the treatment-decision pathway for node-negative and micrometastatic disease

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• Tumor 0.6-1.0 cm, moderately or poorly differentiated, intermediate or high grade, or vascular invasion

• Tumor > 1 cm with favorable or unfavorable pathologic features

Consider Oncotyp

e DX

Hormone receptor-positive, HER2-negative disease

pT1, pT2, or pT3 and pN1mi

No test

RS < 18

RS 18-30

RS ≥ 31

Adjuvant endocrine therapy

± adjuvant chemotherapy

Adjuvant endocrine therapy

endocrine therapy ± adjuvant chemotherapy

Adjuvant endocrine therapy

+ adjuvant chemotherapy

THANK YOU